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Pharmacogn J. 2020; 12(6): 1206-1217 A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article www.phcogj.com

Caesalpinia sappan L. Wood is a Potential Source of Natural Phosphodiesterase-1 Inhibitors

Helmi1, Nanang Fakhrudin2,*, Arief Nurrochmad3, Ari Sudarmanto4, Zullies Ikawati3

ABSTRACT Introduction: A decrease in cAMP and cGMP levels in the brain is linked to human cognitive problems. The degradation of cellular cAMP and cGMP is attributed to phosphodiesterases (PDEs), which constitute a superfamily of enzymes. The inhibition of PDE1 is a promising Helmi1, Nanang Fakhrudin2,*, mechanism to increase cAMP and cGMP levels associated with cognitive disorders. Arief Nurrochmad3, Ari Caesalpinia sappan L. (CS) wood is a natural coloring agent usually consumed as a traditional Sudarmanto4, Zullies Ikawati3 refreshment or drink by people in Yogyakarta, Indonesia. However, scientific evidence regarding the inhibitory activity of CS wood against PDE1 has yet to be obtained. This study aimed to 1Faculty of Pharmacy, Universitas Gadjah investigate the potency of CS wood as a PDE1 inhibitor. Methods: The ethanol extract of CS Mada, Sekip Utara, Yogyakarta 55281, wood and its fractions were evaluated in vitro by using a cyclic nucleotide phosphodiesterase INDONESIA. 2Department of Pharmaceutical Biology, assay kit. The presence of brazilin in the extract and fractions was analyzed by thin-layer Faculty of Pharmacy, Universitas Gadjah chromatography. In silico assay was performed using MOE software to obtain insights into the Mada, Sekip Utara, Yogyakarta 55281, interaction between compounds in the CS wood and the enzyme. Results: Ethanol extract INDONESIA. and ethyl acetate soluble fraction effectively inhibited the PDE1 activity. Interestingly, brazilin, 3Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas the major compound in CS wood, also exhibited a potent inhibitory effect on the enzyme. Gadjah Mada, Sekip Utara, Yogyakarta The in silico assay revealed that the interaction between tetraacetylbrazilin and brazilin with 55281, INDONESIA. the PDE1B active site involved hydrogen bonding and π–π interactions. Conclusion: Ethanol 4 Department of Pharmaceutical Chemistry, extract, ethyl acetate soluble fraction, and brazilin inhibited the PDE1 activity. CS wood and its Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, chemical constituent could be developed as natural cognitive enhancers. INDONESIA. Key words: Brazilin, Caesalpinia sappan L., Phosphodiesterase inhibitor, Herbal cognitive enhancer, Phytotheraphy, Tetraacetylbrazilin. Correspondence Nanang Fakhrudin Department of Pharmaceutical Biology, INTRODUCTION in CI because the inhibition of PDE1 improves Faculty of Pharmacy, Universitas Gadjah cognitive abilities.8,9,15-17 Thus, the use of synthetic Mada, Sekip Utara, Yogyakarta 55281, Cognitive impairment (CI) is a neurodegenerative INDONESIA. PDE1 inhibitors (e.g., vinpocetine) effectively disorder that disrupts memory and learning increases memory and learning abilities in patients E-mail: [email protected] 1,2 abilities. Memory and learning are important with dementia. Unfortunately, vinpocetine exerts History aspects of human cognitive functions regulated by undesirable side effects, such as stomach pain, • Submission Date: 22-05-2020; intercellular cyclic nucleotides and cyclic adenosine nausea, sleep disturbances, headache, dizziness, • Review completed: 29-06-2020; monophosphate/cyclic guanosine monophosphate nervousness, and flushing. These effects have become • Accepted Date: 06-07-2020. (cAMP/cGMP). cAMP/cGMP is a second more profound among the elderly.18-20 Therefore, the DOI : 10.5530/pj.2020.12.169 messenger that plays a key role in the modulation development of PDE1 inhibitors is still a promising of human neuronal functions. In a clinical context, Article Available online approach to combat CI. PDE1 inhibitors may be cAMP/cGMP levels in the brain of patients with http://www.phcogj.com/v12/i6 3,4 obtained from medicinal plants because they have CI decrease. The enzyme responsible for the been considered a basis for discovering bioactive Copyright degradation of cAMP/cGMP is phosphodiesterase molecules.6,10,21 © 2020 Phcogj.Com. This is an open- (PDE). PDE is a superfamily of enzymes grouped access article distributed under the terms Secang (Caesalpinia sappan L.; CS) is an Indonesian of the Creative Commons Attribution 4.0 into 11 subfamilies (PDE1–PDE11) based on their International license. substrate specificity. Among these subfamilies, medicinal plant widely used as a natural red pigment PDE1 is the most abundantly expressed in the in several herbal medicines. It is also the main human brain.5 ingredient of “Wedang Uwuh,” a traditional drink The inhibition of PDE1 causes the upregulation composed of CS wood, clove leaf, and ginger, and it is commonly consumed as a stamina enhancer, of intercellular cAMP/cGMP levels in the brain. 22 Consequently, cognitive performance increases.5-10 body warmer, and refreshment drink. Previous These second messengers are required for the studies reported that CS wood has antioxidant, expression of brain-derived neurotrophic factor antiinflammation, antibacterial, anticancer, (BDNF) via the protein kinase A/G (PKA/G)- antiacne, vasodilator, hepatoprotector, antidiarrheal, 23,24 cAMP response element binding (CREB) signaling aphrodisiac, and antidiabetic activities. However, pathway. BDNF is an important protein for the its cognitive enhancer activity via the inhibition of proliferation, differentiation, and plasticity of PDE1 has yet to be scientifically reported. Here, we synapses in the regulation and maintenance evaluated the inhibitory activity of CS wood extract, of cognitive function, especially learning and fractions, and brazilin (as the major compound in memory.11-14 PDE1 is a promising molecular target CS wood) against PDE1 in vitro and predicted their

Cite this article: Helmi, Fakhrudin N, Nurrochmad A, Sudarmanto A, Ikawati Z. Caesalpinia sappan L. Wood is a Potential Source of Natural Phosphodiesterase-1 Inhibitors. Pharmacogn Phcogj.com J. 2020;12(6):1206-17.

1206 Pharmacognosy Journal, Vol 12, Issue 6, Nov-Dec, 2020 Helmi, et al.: Caesalpinia sappan L. Wood is a Potential Source of Natural Phosphodiesterase-1 Inhibitors molecular interaction in an in silico model. This study contributed to chloroform:acetone:formic acid (4:2:0.5 v/v) as stationary and mobile the development of cognitive enhancer agents from natural origins. phases, respectively. Compound spots were observed under ultraviolet (UV) light at 254 and 366 nm and visible light after derivatization MATERIALS AND METHODS by using a Cerium sulfate spray reagent. The presence of the major Plant materials compound was confirmed with commercially available brazilin (Chengdu Biopurity, China) as a reference compound. CS wood was collected from Imogiri, Bantul, D. I. Yogyakarta, and the plant was authenticated by a botanist at the Department of In silico experiment Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah A molecular docking experiment was performed with Molecular Mada. The CS wood bark was initially washed and then dried in an Operating Environment (MOE) software version 2016.01. A PDE1B oven for 12 h. The dried bark was cut into small pieces and ground isoform (PDB ID: 5W6E) was used with a resolution of 1.90 Å. The using a laboratory grinding mill to produce CS wood powder. isoform used as a protein target in this docking experiment was Chemicals associated with spatial and contextual memory regulatory function in the hippocampus.25 Target protein crystals were acquired from Protein The following materials were used in this study: cyclic nucleotide Data Bank (RCSB PDB, https://www.rcsb.org/). The 3D structures of phosphodiesterase assay kit (Enzo®, Enzo Life Science, USA; cat. 28 tested compounds were obtained from SMILES ID in PubChem number: BML-AK800); methanol, ethanol, ethyl acetate, chloroform, (https://www.pubchem.ncbi.nlm.nih.gov/). acetone, and thin-layer chromatography (TLC) silica gel 60 F254 (20 cm × 20 cm; Merck, Darmstadt, Germany); 3-isobutyl-1-methylxanthine, Ligand and target protein preparation vinpocetine, formic acid, and Cerium(IV)sulfate (Ce(SO4)2; Sigma Target proteins were prepared by adding hydrogen atoms, yielding an Aldrich, USA); and brazilin (Chengdu Biopurity, China). atomic formal charge, and dispensing water molecules and unnecessary CS wood extract and fraction preparation amino acids. Ligands were prepared by minimizing their molecular energy with MMFF94 until the molecular conformation with the lowest CS wood powder (600 g) was macerated with 600 mL of ethanol for 24 energy was obtained. h and filtered. The macerate was concentrated using a rotary evaporator (STUART® RE300) at 50 °C and left at room temperature until dryness. Molecular docking validation The ethanol extract (50 g) was dissolved with warm destilled water and Molecular docking was validated by redocking the target protein with partitioned with ethyl acetate (1:10 v/v) to produce ethyl acetate soluble its native ligands. PDE1B was docked with PF04677490, a native ligand, and insoluble fractions. These fractions were evaporated in a rotary in accordance with the following protocol. The ligands were used as the evaporator and left at room temperature until dryness. site, induced fit was set as the refinement method, and Alpha PMI and Tested solution preparation GBVI/WSA dG were used as the algorithm for the scoring function. This process was set at 310 K and pH 7.4 and run 10 times. The target The dried extract and fractions (each 25 mg) were dissolved in 50 mL protein was assumed valid and ready for molecular docking if the of distilled water and homogenized with an ultrasonic bath (Branson® RMSD value was <2 Å and if it had a good protein–ligand complex B-220) for 15 min. These solutions were diluted to obtain the final conformation. concentration of 500 µg/mL for in vitro assay. Brazilin (5.2 mg) and vinpocetine (5.0 mg) were dissolved in 5 mL of distilled water and Molecular docking of the tested compounds diluted to prepare a final concentration of 11.45 and 14.02 µg/mL, Molecular docking was conducted on 27 compounds previously respectively. reported as chemical constituents of CS wood. Vinpocetine, a selective PDE1 inhibition assay PDE1 inhibitor, was used as a positive control. Docking was conducted in accordance with the protocol from the previous validation. Docking The PDE1 inhibition assay of the extract and fractions of CS wood was run at human physiological temperature and pH to obtain binding were investigated using a cyclic nucleotide phophodiesterase assay kit free energy (∆G bind; kcal/mol) as the test parameter in this study. (ENZO Life Science® BML-AK800). The first mixture to be prepared was composed of 20 µL of 0.5 mM 3ʹ,5ʹ-cyclic adenosine monophosphate Statistical analysis (cAMP), 10 µL of 5 kU/µL 5ʹ-nucleotidase, 5 µL of 10 mM PDE assay Data were analyzed through one-way ANOVA and post hoc analysis buffer, 10 µL of 100 µg/mL extract and fraction solution, 11.45 µg/mL with Bonferroni tests. Significance level was determined at p < 0.05 by brazilin, and 14.02 µg/mL vinpocetine. Then, 5 µL of 4 mU/µL PDE1 was using IBM SPSS Statistics version 21. added to the first mixture and incubated at 30 °C for 30 min. Biomol® Green Reagent (100 µL) was added to terminate the enzymatic reaction. RESULTS AND DISCUSSION After 30 min of incubation, absorbance was measured at 620 nm in a PDE belongs to a group of hydrolytic enzymes that catalyze the microplate reader (Corona® SH-1000). The serial concentrations of 3, 2, hydrolysis of a phosphodiester bond on 3ʹ,5ʹ-cyclic adenosine 1.5, 1, 0.75, 0.5, and 0.25 nmol 5ʹ-adenosine monophosphate (5ʹ-AMP) monophosphate/3ʹ,5ʹ-cyclic guanosine monophosphate (cAMP/cGMP) were used to generate a standard curve. An assay buffer was utilized as ʹ ʹ a negative control. The 5ʹ-AMP level in each experiment was calculated into 5 -adenosine monophosphate/5 -guanosine monophosphate through linear regression obtained from the standard curve of 5ʹ-AMP. (AMP/GMP). This enzyme is important in various physiological The percentage of PDE1 inhibition was calculated with the following functions, including memory function. It is also associated with formula: [(A − B) / A] × 100%, where A and B represent the means of cognitive disorders in human. PDE1 is highly expressed in the cortex, 5ʹ-AMP level of the negative control and the tested sample, respectively. hippocampus, olfactory bulbs, and brain stem, and these regions participate in the regulation of cognitive functions, such as learning and TLC analysis memory.7 Thus, PDE is a potential target for combating CI. The presence of brazilin in the extract and fractions (10 mg/mL In this study, the activity of CS wood extract and its fractions was solution) was analyzed through TLC by using silica gel F254 and evaluated. Vinpocetine, a selective PDE1 inhibitor derived from Vinca

Pharmacognosy Journal, Vol 12, Issue 6, Nov-Dec, 2020 1207 Helmi, et al.: Caesalpinia sappan L. Wood is a Potential Source of Natural Phosphodiesterase-1 Inhibitors rosea alkaloid, was used as a positive control.18 The in vitro enzymatic assay revealed that the CS extract and the ethyl acetate soluble fractions elicited a significant inhibitory effect on PDE-1 (Figure 1). No activity was observed in the ethyl acetate insoluble fraction. As expected, vinpocetine showed a potent inhibitory effect on the PDE1 activity, suggesting that this bioassay was sensitive to the presence of PDE1 inhibitor. Interestingly, the inhibitory activity of the extract was stronger (63.48%) than that of the ethyl acetate soluble fraction (54.78%) tested at 100 µg/mL. A potential synergistic effect might contribute to this activity.26 However, further studies might be required to clarify this synergism. Figure 1: Inhibitory activity of the CS extract, fractions, and brazilin against PDE1. The assay was conducted using a PDE1 inhibition assay CS wood contains brazilin as a major compound and other various kit (cyclic nucleotide phosphodiesterase assay kit). The extract and the types of compounds, such as flavones, homoisoflavonoids, chalcones, fractions were tested at 100 µg/ml, whereas brazilin was examined at 40 xanthone, and dibenzoxocins.24,27,28 In line with previous studies, our µM or 11.45 µg/mL. Data were analyzed with one-way ANOVA followed phytochemical investigation via TLC analysis found that the CS wood by Bonferroni post hoc test. Data were presented as mean ± SEM (*p < 0.05; n.s: not significant compared with the solvent control). extract and the ethyl acetate soluble fraction also contain brazilin. Brazilin appeared as a brown spot observed under visible light after Cerium(IV)sulfate staining, and the spot has similar color and Rf value with the brazilin reference compound (Figure 2). The in vitro assay involving brazilin revealed that this compound effectively inhibited the PDE1 activity with the percentage of inhibition of 34.90% at a concentration of 11.45 µg/mL. This result indicated that brazilin is an active compound that might contribute to the inhibitory effect of PDE1. Previous studies reported that CS wood contains brazilein, brazilide A, tetraacetylbrazilin, brazilane, 3-O-methylbrazilin, neuprotosappanin, protosappanin E1, and hematoxylin.24,28 Some of these compounds have structural similarity, so they may be active PDE1 inhibitors as shown by brazilin. Based on that, an in silico approach was applied to predict the interaction between 27 CS wood chemical constituents and PDE1B. In silico approach was conducted using MOE version 2016.01 software. The selected target protein was validated before a molecular docking study was performed. Molecular docking was analyzed using Figure 2: TLC profile of the extract, fractions, and brazilin. (1) Brazilin (2), the acquired root mean square deviation (RMSD) values. RMSD is ethanol extract of CS wood, (3) ethyl acetate soluble fraction, and (4) ethyl a parameter that represents the reproducibility of a target protein– acetate insoluble fraction. TLC was conducted on a stationary phase of native ligand complex in the formation of a suitable and appropriate silica gel F254 by using the mobile phase of chloroform:acetone:formic conformation. An appropriate RMSD value is less than 2 Å, but its ideal acid (4:2:0.5 v/v). The TLC plate was visualized under visible light after it value is less than 1 Å. The superimposition and RMSD value of protein was sprayed with Cerium sulfate (a) and exposed to UV light at 254 (b) target redocking with its native ligand, PF04677490, is shown in Figure and 366 nm (c). 3. The calculated ∆G bind scores of the tested compounds are presented in Table 1. ∆G bind is an important parameter because it represents the receptor– ligand binding strength between PDE1B and compounds in CS wood. A low ∆G bind score also indicates the stability and strength of the interaction between an enzyme (e.g., PDE1B) and its ligands. These factors contribute to the resulting pharmacological effects. In Table 1, 22 (compound numbers 2–23) of the 28 tested compounds in CS wood were predicted to inhibit the PDE1B activity. Vinpocetine, as the reference drug, was predicted to interact with the enzyme, with ∆G bind of −5.438 kcal/mol. Among the tested compounds, tetraacetylbrazilin is predicted to have the strongest interaction with PDE1B, whereas protosappanin B has the weakest interaction; they had ∆G bind of −8.691 and −5.498 kcal/mol, respectively. ∆G bind of protosappanin B is equal to that of vinpocetine and almost equal to brazilin, the major compound in CS wood (∆G bind of −5.823 kcal/ mol). Protosappanin D, protosappanin E1, caesalpin J, caesalpinin P, and 2,4,5-trihydroxybenzaldehyde were predicted as inactive because they demonstrated ∆G bind lower than that of vinpocetine (−5.438 kcal/mol). The interaction of tetraacetylbrazilin and brazilin with Figure 3: Superimposition of the redocked PF0467749, the native ligand compound of PDE1B. The poses show an overlapping or a similar PDE1B is illustrated in Figure 4. interaction before (green) and after (grey) redocking with amino acid In Figure 4, tetraacetylbrazilin showed two π–π interactions with residues on the PDE1B active site. The RMSD score was 0.27 Å (<1 Å; PDB the enzyme; that is, their interaction involves the benzene rings of ID: 5W6E).

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Figure 4: Interaction of tetraacetylbrazilin (1) and brazilin (2) with amino acid residues on the PDE1B active site. The interaction is illustrated in 3D (a) and 2D (b) models by using MOE ver. 2016.01.

Table 1: Binding free energy (∆G bind) scores of compounds in CS wood (2-28) and vinpocetine as a positive control (1). Numbers 2–28 correspond to the strength of the interaction between PDE1B and the respective compound. No. Compounds ∆G bind (kcal/mol)

O O

1 N −5.438 H NH+

Vinpocetine

O O O O O 2 O −8.691 O O

O Tetraacetylbrazilin

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HO O OH

3 OH −7.081 OH O Sappanone B

HO OH

O OH OH 4 −7.007 HO

HO O

OH Neuprotosappanin

5 −6.960 HO O OH 3-Deoxysappanchalcone

HO O OH

6 OH −6.779 O 3-Deoxysappanone B

O HO 7 −6.723 HO HO OH Butein

O OH 8 −6.717 HO O OH Sappanchalcone

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9 −6.690 O O

OH 3-O-methylbrazilin

OH HO O HO OH

10 −6.596 OH O Caesalpiniaphenol G

HO O OH

11 OH −6.549 O Sappanon A

HO O OH OH 12 OH −6.435 OH Episappanol

13 O −6.425 O O O OH Brazilane

14 HO −6.246 O

HO Protosappanin A (Sappanol B)

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15 −6.141 HO O

OH Brazilein

16 O O O −6.085 O O OH Brazilide A

17 −5,823 HO O

HO OH Brazilin

18 −5.796 O HO O Urolitin C

O OH 19 −5.737 OH Caesalpiniaphenol H

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HO OH 20 −5.630 HO O OH Hematoxylin

HO 21 O −5.604

HO O HO Protosappanin C

O OH HO 22 −5.524 O Euxanthone

HO 23 O −5.498

HO OH HO Protosappanin B

HO OH HO OH

OH O 24 −5.106

HO O O O OH HO Protosappanin D

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OH HO OH

O O HO O 25 −5.092 HO O

OH OH Protosappanin E1

26 HO −4.924 OO

HO OH Caesalpin J

HO OH

27 O −4.593 HO 2,4,5-Trihydroxybenzaldehyde

O O O O

28 O −4.384 OH

Caesalpinin P tetraacetylbrazilin and the two benzene rings of phenylalanine-A446 inhibitory activity of CS wood extract might be related to the synergistic (PheA446) and phenylalanine-B446 (PheB446) residues of the enzyme effect of its constituent involving at least 22 compounds. However, their with a similar bond length of 3.66 Å. Interestingly, brazilin showed two effectiveness in synergism must be confirmed in relevant bioassays. distinct chemical interactions with the PDE1B active site. In particular, These findings indicated that CS wood could be a potential source of a π–π interaction occurs between the benzene ring of brazilin and a natural cognitive enhancer agent for combating memory disorders. the benzene ring of the Phe446 with a bond length of 3.66 Å, and a hydrogen donor interaction takes place between the hydroxyl group CONCLUSION (O-23) and the nitrogen of histidin-234 (His234) residue with a bond In this study, CS wood extract, ethyl acetate fractions, and brazilin length of 3.18 Å. This interaction is consistent with a previous study.29 as the major compound of CS wood inhibited the PDE1 activity in Our in silico study showed that at least 22 constituent compounds of the enzymatic in vitro assay. However, the activity of the extract was CS wood extract might interact with PDE1, as indicated by low ∆G stronger than that of the fraction. Our in silico approach revealed that bind scores docked with PDE1B. This finding suggested that the PDE1 PDE1B interacted with at least 22 constituents of CS wood.

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13. Nair A, Vaidya VA. Cyclic AMP response element binding protein and brain- ACKNOWLEDGMENT derived neurotrophic factor: Molecules that modulate our mood? Journal of The authors would like to express their gratitude to the Ministry of Biosciences. 2006;31:423-34. Research, Technology, and Higher Education (KEMENRISTEKDIKTI) 14. Keravis T, Lugnier C. Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in through the scholarship of the Program Magister menuju Doktor various diseases and perspectives for future therapeutic developments: PDEs: untuk Sarjana Unggul (PMDSU) with the contract number 3167/UN1. intracellular network targets for diseases. British Journal of Pharmacology. DITLIT/DIT-LIT/LT/2020 for the financial support during the research 2012;165:1288. and preparation of this manuscript. 15. Blokland A, Menniti FS, Prickaerts J. PDE Inhibition and cognition enhancement. 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GRAPHICAL ABSTRACT

ABOUT AUTHORS

apt. Helmi, S.Farm. apt. Helmi, S.Farm. is a Ph.D Candidate at the Doctoral Degree Program in the Pharmaceutical Science-Technology, Faculty of Pharmacy, Universitas Gadjah Mada. His graduate research is on herbal medicines for cognitive enhancer.

Dr.rer.nat. apt. Nanang Fakhrudin, M.Si. Dr.rer.nat. apt. Nanang Fakhrudin, M.Si. is a lecturer and researcher at Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada. He is doing researches in phytochemistry and biological activities of medicinal plants and bioactive compounds, including bioactivites screening.

Dr. apt. Arief Nurrochmad, M.Si., M.Sc. Dr. apt. Arief Nurrochmad, M.Si., M.Sc. is a lecturer and researcher at Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada. He is actively doing researches in pharmacology and toxicology. His research interest are clinical toxicology, molecular pharmacology and toxicology, neuropharmacology and pharamcotherapy of degenerative disease. His invention in derivatives of benzylidene cyclohexanone, benzylidene cyclopentanone and benzylidene acetone and therapeutic uses thereof was granted as US patent in 2004.

1216 Pharmacognosy Journal, Vol 12, Issue 6, Nov-Dec, 2020 Helmi, et al.: Caesalpinia sappan L. Wood is a Potential Source of Natural Phosphodiesterase-1 Inhibitors

Dr. B. S. Ari Sudarmanto, M.Si. Dr. B. S. Ari Sudarmanto, M.Si. is a lecturer and researcher at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada. His major expertize is in pharmaceutical chemistry with the research of interest in computational chemistry and medicinal chemistry.

Prof. Dr. apt. Zullies Ikawati Prof. Zullies Ikawati, PhD, Pharm is academician with specialization in Pharmacology and Clinical Pharmacy in Faculty of Pharmacy Universitas Gadjah Mada. She earned PhD degree in Pharmacology from Ehime University Japan in 2001. She is actively doing research in the field of pharmacology, clinical pharmacy, and pharmacogenomics. She conducted several clinical trials of herbal medicine products for several indication, such as diabetes mellitus, hyperlipidemia, and common cold. She has published many articles in international journals, with interest of pharmacology, clinical pharmacy and pharmacogenomics.

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